NF-κB in the mechanism of brain edema in acute liver failure: studies in transgenic mice.

Department of Pathology, University of Miami School of Medicine, Miami, FL 33101, USA.

Abstract

Astrocyte swelling and brain edema are major complications of the acute form of hepatic encephalopathy (acute liver failure, ALF). While elevated brain ammonia level is a well-known etiological factor in ALF, the mechanism by which ammonia brings about astrocyte swelling is not well understood. We recently found that astrocyte cultures exposed to ammonia activated nuclear factor-κB (NF-κB), and that pharmacological inhibition of such activation led to a reduction in astrocyte swelling. Although these findings suggest the involvement of NF-κB in astrocyte swelling in vitro, it is not known whether NF-κB contributes to the development of brain edema in ALF in vivo. Furthermore, pharmacological agents used to inhibit NF-κB may have non-specific effects. Accordingly, we used transgenic (Tg) mice that have a functional inactivation of astrocytic NF-κB and examined whether these mice are resistant to ALF-associated brain edema. ALF was induced in mice by treatment with the hepatotoxin thioacetamide (TAA). Wild type (WT) mice treated with TAA showed a significant increase in brain water content (1.65%) along with prominent astrocyte swelling and spongiosis of the neuropil, consistent with the presence of cytotoxic edema. These changes were not observed in Tg mice treated with TAA. Additionally, WT mice with ALF showed an increase in inducible nitric oxide synthase (iNOS) immunoreactivity in astrocytes from WT mice treated with TAA (iNOS is known to be activated by NF-κB and to contribute to cell swelling). By contrast, Tg mice treated with TAA did not exhibit brain edema, histological changes nor an increase in iNOS immunoreactivity. We also examined astrocytes cultures derived from Tg mice to determine whether these cells exhibit a lesser degree of swelling and cytopathological changes following exposure to ammonia. Astrocyte cultures derived from Tg mice showed no cell swelling nor morphological abnormalities when exposed to ammonia for 24h. By contrast, ammonia significantly increased cell swelling (31.7%) in cultured astrocytes from WT mice and displayed cytological abnormalities. Moreover, we observed a lesser increment in iNOS and NADPH oxidase activity (the latter is also known to be activated by NF-κB and to contribute to astrocyte swelling) in astrocyte cultures from Tg mice treated with ammonia, as compared to ammonia-treated WT mice astrocytes. These findings strongly suggest that activation of NF-κB is a critical factor in the development of astrocyte swelling/brain edema in ALF.

Light microscopy (Giemsa stain) of cultured astrocytes from WT and Tg mice treated with ammonia. A. Astrocytes from control Tg mice show a monolayered sheet of cells possessing a moderate amount of light-staining cytoplasm, fine cytoplasmic processes and round to oval nuclei with evenly distributed chromatin. B. Astrocytes from Tg mice treated with NH4Cl (5 mM) for 72 h show no significant cytopathological changes as compared to Tg control. C. Astrocytes from control WT mice are similar to control Tg mice astrocytes. D. Cultured astrocytes from WT mice treated with NH4Cl (5 mM) for 72 h display slightly shrunken nuclei along with a more prominent and darker cytoplasm, which has a more stellated appearance. Many cells contain numerous vacuoles (long arrows) as well as occasional dense bodies (short arrows). E. WT mice astrocytes exposed to ammonia display similar feature as in D. The cytoplasm, however, is darker and the vacuolization is more intense. All x300.